Infusion of C1 inhibitor (C1INH) protects mice, dogs, rabbits and baboons from gram negative bacterial sepsis and shock. Protection has been assumed to result from inhibition of activation of the complement and/or contact systems. Because C1INH is so effective, we hypothesized that it might bind to LPS, and that this interaction might interfere with the ability of LPS to activate macrophages. Preliminary studies support this hypothesis. Specific Aim 1 will analyze the C1INH-LPS interaction. The binding site will be defined using recombinant mutated C1INH proteins, which will be analyzed for the ability to bind to LPS (ELISA and surface plasmon resonance), to inhibit binding of LPS to, and activation of, macrophages. The ability of C1 INH to protect mice from endotoxin shock will be directly compared with that of other endotoxin antagonists. Specific Aim 2 will define the role of C1INH in protection from LPS-induced endothelial injury. To analyze the effect of C1INH on LPS-induced vascular permeability, endothelial cell cytoskeletal alterations and cell barrier dysfunction will be characterized, as will the ability of C1INH, C1INH mutants and the amino terminal domain to reverse LPS-mediated increased vascular permeability. Specific Aim 3 will characterize the clearance and organ localization of C1INH-LPS complexes in mice. In addition, the internalization and degradation of C1INH-LPS complexes by different cell types will be analyzed in vitro. The effect of C1INH on LPS tolerance in mice (induced by low doses of LPS) will be determined. Lastly, because sepsis models are more biologically relevant to human disease and because some agents that are protective in endotoxin shock models may not be protective (or may even be harmful) in sepsis, Specific Aim 4 will analyze the role of C1INH and C1 INH variants in protection in the cecal ligation/puncture model in mice. These studies will lead to improved understanding of the role played by C1INH in the cbrresistance to gram negative sepsis and endotoxin shock, and may lead to new therapies for these diseases.